Myeloid derived suppressor cells (MDSC) are present in virtually all cancer patients, where they are induced and activated by inflammation. Because they are a major obstacle to immunotherapy of cancer, it is imperative to understand how these cells mediate their effects and communicate within the tumor microenvironment. The long-term objective of this collaborative program is to identify the molecules that MDSCs use to hijack the immune system and to use this information to re-program or neutralize MDSC. Preliminary evidence indicates that this intercellular communication is facilitated by exosomes, nanoscale membrane-bound vesicles shed by MDSC. We have identified several proteins in MDSC exosomes that have significant physiologic effects on other MDSC, and on macrophages, another population of myeloid cells that promote tumor growth. Preliminary experiments also indicate that proteins conjugated with ubiquitin chains are readily enriched from exosomes shed by MDSC. Protein modification by ubiquitination is a powerful signal, which is known to direct proteins to diverse cellular fates according to structural variations in te massive polyubiquitin tags. This multi-PI study will test the hypothesis that ubiquitinated protein in exosomes shed by MDSC and macrophages contribute to immune suppression. We will ask how ubiquitination marks cellular proteins for export as exosome cargo, and if ubiquitination is altered by inflammatory stimulation. Mass spectrometry has provided most of the information presently available about sites of ubiquitin conjugation on targeted proteins; however characterization of the linkages within ubiquitin chains remains a significant analytical challenge We will use novel synthetic methods to prepare polyubiquitins and proteins conjugated with polyubiquitins as standard reference compounds. We will use these standards to develop and test a proteomic strategy to characterize connection sites and their sequence in ubiquitin chains. This novel customized strategy, incorporating Asp-selective proteolysis, linkage-specific antibodies, LC-MS/MS and bioinformatics, will then be applied to characterize ubiquitin conjugates from exosomes shed by MDSC and macrophages. We will focus initially on chains with isopeptide bonds at K63, K48 and K11. These are considered to occur most frequently, have known functional correlations, and can be addressed by linkage-specific antibodies. Biological activity of selected ubiquitinated and other exosomal proteins will be validated by determining their ability to enhance pro-tumor activity and immune suppressive potency.